IL-10-Secreting CD8+ T Cells Specific for Human Cytomegalovirus (HCMV): Generation, Maintenance and Phenotype

Abstract

HCMV-specific CD8⁺ T-cells are potent anti-viral effector cells in HCMV infected individuals, but evidence from other viral infections suggests that CD8⁺ T-cells can also produce the immunomodulatory cytokine IL-10. In this work we show that there are HCMV-specific IL-10 CD8⁺ T-cell responses in a cohort of individuals aged 23-76 years of age, predominantly directed against the HCMV proteins known to be expressed during latent infections as well as towards the proteins US3 and pp71. The analysis of HCMV-specific responses established during primary infection has shown that the IL-10 responses to US3 and pp71 HCMV proteins are detectable in the first weeks post infection, but not the responses to latency-associated proteins, and this IL-10 response is produced by both CD8⁺ and CD4⁺ T-cells. Phenotyping studies of HCMV-specific IL-10⁺ CD8⁺ T-cells show that these are CD45RA⁺ effector memory cells and co-express CD28 and CD57, however, the expression of the inhibitory receptor PD-1 varied from 90% to 30% between donors. In this study we have described for the first time the HCMV-specific IL-10 CD8⁺ T-cell responses and have demonstrated their broad specificity and the potential immune modulatory role of the immune response to HCMV latent carriage and periodic reactivation.

Keywords: CD8+ T cells; IL-10 secretion; T regulatory cells; human cytomegalovirus (HCMV); immunomodulation.

Figure 1

HCMV-specific CD8⁺ T cells produce IL-10 in response to HCMV protein stimulation. The IL-10-secreting CD8⁺ T cell response to 10 HCMV proteins UL138, US28, LUNA, UL144, vIL-10 (latency-associated proteins, dark blue colored points), pp71, US3, pp65, IE2, IE1 (lytically expressed proteins, peach colored points) and the positive control was measured in a cohort of 46 HCMV-seropositive donors using a IFNγ and IL-10 dual FluoroSpot technique. The results were converted to sfu/million T cells with background counts subtracted and the positive response threshold of 100 sfu/million is indicated (dashed line) on the graph (A) with the geo-mean of each protein response shown as a line. The IL-10 response to the lytically expressed HCMV proteins (B) and the latency-associated proteins (C) is shown as a correlation of donor age with the cumulative size of the donor response. The breadth of the IL-10 CD8⁺ response (the number of HCMV proteins to which each donor had an IL-10 response above-threshold) was enumerated and plotted by age (D). The correlation of the response with age was analyzed using the Spearman rank correlation (Spearman r_s with 95% confidence intervals (CI) and p value are indicated with the line of best fit (solid) and 95% CI (dotted lines) also shown on the graphs). The proportion of donors responding to one or more of the HCMV proteins are summarized as a pie chart with the key to segments shown (E).

Figure 2

Dominant cytokine response to HCMV protein stimulation. The dominant cytokine response to HCMV protein stimulation of CD8⁺ T cells was calculated. A dominant response was defined as over 55% of the total CD8⁺ T cell-positive response; the number of donors generating a dominant IFNγ response (navy bars) or IL-10 response (peach bars) or an equivocal response (green bars) are shown as stacked bars for the 10 HCMV proteins. Also indicated are the total number of responding donors (either cytokine) to each of the 10 HCMV proteins.

Figure 3

PBMC were stimulated overnight with HCMV peptide pools in the presence of monensin. The identification of HCMV-specific CD8⁺ T cell-positive responses was determined by the expression of 4-1BB and/or CD69 upregulation above background and the percentage of IL-10 and IFNγ response in the positive cells measured. The background-corrected percentages of 4-1BB⁺ and/or CD69⁺ CD8⁺ T cells to each HCMV peptide pool are shown with the positive threshold of 0.25% (dotted line) indicated (A). The percentage of IL-10 (B) and IFNγ (C) secretion of HCMV-specific CD8⁺ T cells from 17 donors to the seven HCMV proteins pp65, IE (peach filled shapes, lytically expressed proteins), UL144, US28, UL138, LUNA and vIL-10 (dark blue filled shapes, latency-associated proteins) are shown.

Figure 4

HCMV-specific IL-10 responses arise early in primary infection. A cohort of seven D⁺R⁻ transplant patients who had primary HCMV infection post transplantation was recruited by the Academisch Medisch Centrum (AMC) Amsterdam. PBMC were collected at multiple time points after transplantation and analyzed for T cell secretion of IFNγ and IL-10 by FluoroSpot in response to pooled HCMV protein stimulations. The background corrected responses of the total PBMC from the seven patients are illustrated showing the IFNγ and IL-10 T cell responses over the weeks post-transplant for IE1 and IE2 stimulation (A), UL138, US28, LUNA and vIL-10 (latency associated proteins) stimulation and (B) pp71 and US3 stimulation, with (C) the positive response threshold of 100 sfu/million CD3⁺ T cells indicated (dotted line).

Figure 5

HCMV-specific IL-10 responses that arise during primary infection are produced by both CD8⁺ and CD4⁺ T cells. Four PBMC samples from D+R- patients 352 (A–C) and 574 (D–F) post-transplant time course were depleted of CD4⁺ or CD8⁺ T cells to assess which T cell subset is producing both IFNγ and IL-10 in response to HCMV-protein stimulation. The IFNγ and IL-10 CD8 (circle points) and CD4 (triangle points) T cell responses of both patients to the positive control (A,D), IE1 and IE2 proteins, (B,E) pp71 and US3 proteins (C,F) stimulation are shown, with the positive response threshold of 100 sfu/million CD3+ T cells indicated (dotted line). The virus load across the time course for each patient is shown (pink line).

Figure 6

Characterization of HCMV latency-associated protein-specific CD8⁺ T cells secreting IL-10. PBMC were stimulated for 6 h with HCMV peptide pools and IL-10 secretion detected by the use of an IL-10-secretion assay kit. Additionally, HCMV-specific CD8⁺ T cell responses were identified by the upregulation of 4-1BB on the cell surface. The phenotype markers of T cell memory subsets and the differentiation status of CD27, CD45RA, CD28 and CD57 were included alongside PD-1 and CD39 markers associated with CD8⁺ IL-10-secreting T cells. Acquired data from each donor was analyzed using dimensionality reduction (tSNE, Flowjo 10.8.1) and clustering analysis (FlowSOM plugin, Flowjo). A summary of the FlowSOM population clustering distribution for unstimulated (US) total CD8s and latency associated protein stimulated (LAT) total CD8, 4-1BB-positive and IL-10-secreting cells is shown for donor CMV319 (A) and CMV332 (D). The dimensionality reduction tSNE plots are shown for total CD8s in both US and LAT conditions with the 4-1BB-expressing (blue overlay), IL-10-secreting (pink overlay) and IL-10 FlowSOM population 5 (purple overlay) also indicated (CMV319, B and CMV332, E). The T cell phenotype of the different populations are also shown with CD28 vs. CD57, CD45RA vs CD27 and PD-1 vs CD39 plots with donor-specific gating shown (CMV319, C and CMV332, F).